scholarly journals In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Yunkun Liu ◽  
Weixin Tao ◽  
Shishi Wen ◽  
Zhengyuan Li ◽  
Anna Yang ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Gene Clusters ◽  
Specific Gene ◽  
Dna Fragments ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Highly Efficient ◽  
Dna Editing

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. IMPORTANCE Recent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.

scholarly journals An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases inMyxococcus xanthusDK1622

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Karla J. Esquilín-Lebrón ◽  
Tye O. Boynton ◽  
Lawrence J. Shimkets ◽  
Michael G. Thomas
Keyword(s):  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

ABSTRACTOne mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 fromMyxococcus xanthusDK1622 for characterization. TheM. xanthusDK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination ofin vivoandin vitroassays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping ofM. xanthusDK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a “universal” MLP for generating functional hybrid NRPSs.IMPORTANCEMbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP fromMyxococcus xanthusDK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a “universal” MLP during the construction of functional hybrid NRPSs.

scholarly journals Zn-dependent bifunctional proteases are responsible for leader peptide processing of class III lanthipeptides

2019 ◽  
Vol 116 (7) ◽  
pp. 2533-2538 ◽  
Author(s):  
Shaoming Chen ◽  
Bing Xu ◽  
Erquan Chen ◽  
Jiaqi Wang ◽  
Jingxia Lu ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Leader Peptide ◽  
Biosynthetic Gene ◽  
Class Iii ◽  
Biosynthetic Gene Clusters ◽  
Peptide Processing ◽  
Leader Peptides ◽  
Biochemical Studies ◽  
Modified Peptides

Lanthipeptides are an important subfamily of ribosomally synthesized and posttranslationally modified peptides, and the removal of their N-terminal leader peptides by a designated protease(s) is a key step during maturation. Whereas proteases for class I and II lanthipeptides are well-characterized, the identity of the protease(s) responsible for class III leader processing remains unclear. Herein, we report that the class III lanthipeptide NAI-112 employs a bifunctional Zn-dependent protease, AplP, with both endo- and aminopeptidase activities to complete leader peptide removal, which is unprecedented in the biosynthesis of lanthipeptides. AplP displays a broad substrate scope in vitro by processing a number of class III leader peptides. Furthermore, our studies reveal that AplP-like proteases exist in the genomes of all class III lanthipeptide-producing strains but are usually located outside the biosynthetic gene clusters. Biochemical studies show that AplP-like proteases are universally responsible for the leader removal of the corresponding lanthipeptides. In addition, AplP-like proteases are phylogenetically correlated with aminopeptidase N from Escherichia coli, and might employ a single active site to catalyze both endo- and aminopeptidyl hydrolysis. These findings solve the long-standing question as to the mechanism of leader peptide processing during class III lanthipeptide biosynthesis, and pave the way for the production and bioengineering of this class of natural products.

scholarly journals A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ye Xu ◽  
Andrew Willems ◽  
Catherine Au-yeung ◽  
Kapil Tahlan ◽  
Justin R. Nodwell
Keyword(s):  
Secondary Metabolites ◽  
Pathogenic Bacteria ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

scholarly journals A widespread family of bacterial gene clusters produces ClpP inhibitors

2021 ◽  
Author(s):  
Gerry Wright ◽  
Elizabeth Culp ◽  
David Sychantha ◽  
Christian Hobson ◽  
Andrew Pawlowski ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Antibiotic Resistance Genes ◽  
Gene Clusters ◽  
Biosynthetic Gene Clusters ◽  
Bacterial Gene ◽  
Clinical Potential ◽  
Novel Scaffold

Abstract Intracellular proteolytic complexes play an essential role in modeling the proteome in both bacteria and eukaryotes. ClpP is the protease subunit of one such highly conserved proteolytic complex that, despite its potential, remains unexploited as a drug target. Here we describe a target-directed genome mining strategy to identify ClpP targeting compounds from the bacterial order Actinomycetales. By searching for biosynthetic gene clusters that contain duplicated copies of ClpP as putative antibiotic resistance genes, we identify a family of ClpP-associated clusters that are widespread across phyla, including environmental and pathogenic bacteria. While numerous bacterial pyrrolizidine alkaloids produced by these gene clusters are known, their connection to ClpP has never been made. We show that these previously characterized molecules do not affect ClpP function but are shunt metabolites derived from the genuine product of these gene clusters, a reactive covalent ClpP inhibitor. Focusing on one such cryptic gene cluster from Streptomyces cattleya DSM 46488, we use heterologous expression to purify the relevant ClpP inhibitor, which we name clipibicyclene. We show in vitro and in vivo that clipibicyclene is a potent covalent inhibitor of ClpP and that cluster-associated ClpPs provide resistance. ClpP inhibition results in antibacterial activity against actinobacteria, including Mycobacterium smegmatis, and inhibition of virulence factor production by Staphylococcus aureus. Finally, we solve the crystal structure of clipibicyclene-modified Escherichia coli ClpP. Clipibicyclene’s discovery deconvolutes the actual function of a family of natural products widespread in nature. It provides a novel scaffold for therapeutic ClpP inhibitor development, making these findings significant from the perspective of their discovery and their clinical potential.

scholarly journals Expansion of gamma-butyrolactone signaling molecule biosynthesis to phosphotriester natural products

2020 ◽  
Author(s):  
Yuta Kudo ◽  
Takayoshi Awakawa ◽  
Yi-Ling Du ◽  
Peter A. Jordan ◽  
Kaitlin E. Creamer ◽  
...  
Keyword(s):  
Natural Products ◽  
Gene Clusters ◽  
Biosynthetic Gene Clusters ◽  
Physiological Processes ◽  
Marine Actinomycete ◽  
Structural Differences ◽  
Species Specific ◽  
Gamma Butyrolactone

AbstractBacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their anti-malarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the saliniphostins and newly identified A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed amongst many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

scholarly journals Identification of genes encoding squalestatin S1 biosynthesis and in vitro production of new squalestatin analogues

2016 ◽  
Vol 52 (41) ◽  
pp. 6777-6780 ◽  
Author(s):  
B. Bonsch ◽  
V. Belt ◽  
C. Bartel ◽  
N. Duensing ◽  
M. Koziol ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Biosynthetic Gene ◽  
In Vitro Production ◽  
Biosynthetic Gene Clusters ◽  
Genes Encoding ◽  
Vitro Production

Biosynthetic gene clusters encoding the production of squalestatin S1 have been discovered and exploited to produce new analogs.

scholarly journals Caries-Associated Biosynthetic Gene Clusters in Streptococcus mutans

2020 ◽  
Vol 99 (8) ◽  
pp. 969-976 ◽  
Author(s):  
S.S. Momeni ◽  
S.M. Beno ◽  
J.L. Baker ◽  
A. Edlund ◽  
T. Ghazal ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Genome Sequencing ◽  
Large Scale ◽  
Biological Activities ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Microbial Diseases ◽  
High Caries Risk

Early childhood caries (ECC) is a chronic disease affecting the oral health of children globally. This disease is multifactorial, but a primary factor is cariogenic microorganisms such as Streptococcus mutans. Biosynthetic gene clusters (BGCs) encode small molecules with diverse biological activities that influence the development of many microbial diseases, including caries. The purpose of this study was to identify BGCs in S. mutans from a high-caries risk study population using whole-genome sequencing and assess their association with ECC. Forty representative S. mutans isolates were selected for genome sequencing from a large-scale epidemiological study of oral microbiology and dental caries in children from a localized Alabama population. A total of 252 BGCs were identified using the antiSMASH BGC-mining tool. Three types of BGCs identified herein—butyrolactone-like, ladderane-like, and butyrolactone-ladderane-like hybrid (BL-BGC)—have not been reported in S. mutans. These 3 BGCs were cross-referenced against public transcriptomics data, and were found to be highly expressed in caries subjects. Furthermore, based on a polymerase chain reaction screening for core BL genes, 93% of children with BL-BGC had ECC. The role of BL-BGC was further investigated by examining cariogenic traits and strain fitness in a deletion mutant using in vitro biofilm models. Deletion of the BL-BGC significantly increased biofilm pH as compared to the parent strain, while other virulence and fitness properties remained unchanged. Intriguingly, BL-BGC containing strains produced more acid, a key cariogenic feature, and less biofilm than the model cariogenic strain S. mutans UA159, suggesting the importance of this BL-BGC in S. mutans–mediated cariogenesity. The structure of any BL-BGC derived metabolites, their functions, and mechanistic connection with acid production remain to be elucidated. Nevertheless, this study is the first to report the clinical significance of a BL-BGC in S. mutans. This study also highlights pangenomic diversity, which is likely to affect phenotype and virulence.

scholarly journals Exonuclease III (XthA) EnforcesIn VivoDNA Cloning ofEscherichia coliTo Create Cohesive Ends

2018 ◽  
Vol 201 (5) ◽  
Author(s):  
Shingo Nozaki ◽  
Hironori Niki
Keyword(s):  
Dna Polymerase ◽  
Dna Fragments ◽  
Dna Polymerase I ◽  
Dna Cloning ◽  
Content Type ◽  
E Coli ◽  
Polymerase I ◽  
Cloning Technology

ABSTRACTEscherichia colihas an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by whichE. coliaccomplishes such cloning is still unknown. In this study, we provide evidence that thein vivocloning ofE. coliis independent of both RecA and RecET recombinases but is dependent on XthA, a 3′ to 5′ exonuclease. Here,in vivocloning ofE. coliby XthA is referred to asin vivoE. colicloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced intoE. colicells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development ofin vivoDNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.IMPORTANCECloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, anin vitrorecombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly,E. colipotentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for thisin vivocloning have realized a high level of usability, comparable to that byin vitrorecombination reactions. However, the mechanism ofin vivocloning is highly controversial. Here, we clarified the fundamental mechanism underlyingin vivocloning and also constructed a strain that was optimized forin vivocloning. Additionally, we streamlined the procedure ofin vivocloning by using a single microcentrifuge tube.

scholarly journals Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

2017 ◽  
Vol 114 (27) ◽  
pp. 7025-7030 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

scholarly journals A Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity

2020 ◽  
Author(s):  
Jack G. Ganley ◽  
Ashmita Pandey ◽  
Kayla Sylvester ◽  
Kuan-Yi Lu ◽  
Maria Toro-Moreno ◽  
...  
Keyword(s):  
Small Molecules ◽  
Genetic Manipulation ◽  
Control Strategies ◽  
Gene Clusters ◽  
Blood Feeding ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Systematic Analysis ◽  
Depth Analysis

ABSTRACTAdvances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito microbiome studies and highlights an understudied source of bioactive small molecules.

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